NIH has identified a need for new technologies to rapidly screen mutant mice to detect and characterize abnormal immune responses, with an emphasis on immune dysfunction associated with autoimmune diseases. The objective of the proposed project will be to develop a novel diagnostic reagent that will bind with high avidity and specificity to antigen-specific T cells. The detection and tracking of T lymphocytes according to their antigen specificity can provide the diagnostic tools to evaluate the immune status of an individual with respect to an infectious disease or autoimmune disorder. Tetramerized, fluorophor-labeled MHC/peptide complexes can bind to antigen-specific T cells, as has been demonstrated in infectious disease models in mice as well as in clinical samples, but this diagnostic system has important limitations: (1) the reagent does not bind to all T cells that can be activated by the particular MHC/peptide complex and (2) each MHC/peptide complex must be analyzed individually. To develop a novel staining reagent that will overcome these limitations, biotinylated MHC/peptide complexes will be bound to fluorescent microspheres and phosphor microspheres as reporter beads, which will allow us to detect many different colors simultaneously, i.e. sets of MHC/peptide complexes can be screened together using the same sample. This will be an important economy for phenotyping mice: the small blood volume of a mouse limits the number of assays that can be performed without killing the animal, so the ability to assay multiple MHC/peptide complexes in one sample will be a major advantage. This opens the possibility of screening large numbers of samples rapidly and efficiently.